The technique of connecting the ends of DNA fragments to prepare arbitrary DNA molecules is a basic technique essential for many experimental procedures in the molecular biological field, including cloning. A current approach most generally used for ligating DNA fragments involves binding blunt-ended DNA molecules or DNA molecules complementary to each other at their sticky ends using DNA ligase. Also, the Cohen-Boyer method (see non-patent document 1) is known as a cloning method based on this approach. This method involves cleaving insert DNA and a vector with their respective restriction enzymes, purifying the fragments, and then ligating the fragments using DNA ligase to prepare the construct of interest, with which E. coli is then transformed, followed by amplification. Since such a DNA ligation method using restriction enzymes and DNA ligase, however, achieves ligation only between DNA molecules prepared in advance to have corresponding ends, the Cohen-Boyer method requires complicated in vitro procedures such as PCR and restriction enzyme treatment for preparing constructs for transformation and further requires a lot of time for selecting E. coli comprising the construct of interest from among colonies of transformed E. coli. For these reasons, the development of more convenient and efficient methods for ligating DNA molecules has been demanded.
In vitro and in vivo homologous recombination methods have been developed as cloning methods using neither restriction enzymes nor DNA ligase as a substitute for the Cohen-Boyer method. The in vitro homologous recombination method connects DNA molecules using specific recombinase and its recognition sequence. For example, the Gateway method of Invitrogen Corp. and the In-Fusion cloning method of Takara Bio Inc. are known. These methods, however, require particular DNA sequences present in both ends of insert DNA and in vectors and therefore, are not excellent in versatility. Alternatively, the known in vivo homologous recombination method uses E. coli (see non-patent documents 2 and 3) or bakery yeast (see non-patent documents 4 and 5). These in vivo homologous recombination methods are based on the fact that E. coli strains caused to express a plurality of phage-derived proteins, or bakery yeast has exceedingly high homologous recombination proficiency. Specifically, insert DNA having, at both ends, additional sequences homologous to a cloning vector and a cloning vector from which a homologous recombination site has been cleaved off with restriction enzymes or the like are prepared, and E. coli or bakery yeast can be transformed therewith to thereby obtain an insert DNA-integrated cloning vector as a result of homologous recombination within the transformed cell (in vivo). This reaction takes place independently of restriction enzyme sites and therefore allows insertion of a DNA fragment to various positions in a vector sequence. Another feature thereof is that cloning efficiency does not vary depending on the length of insert DNA. Both of these methods, however, utilize homologous recombination and thus require adding a sequence homologous to a vector to insert DNA. The in vivo homologous recombination method using E. coli further requires electroporation for obtaining high transformation efficiency and disadvantageously requires, for example, expensive apparatuses or instruments for the procedures. Alternatively, the in vivo homologous recombination method using bakery yeast requires bakery yeast-E. coli shuttle vectors used for transferring cloning vectors to E. coli in the large-scale preparation of DNA. Unfortunately, bakery yeast-E. coli shuttle vectors are not generally used and require time and effort for their preparation.
Alternatively, a known method for constructing and amplifying circular DNA without using restriction enzymes or DNA ligase involves transforming prokaryotic cells with linear DNA obtained by the rolling circle amplification method and reconstructing circular DNA within the transformed cells (see patent document 1). The amplified DNA fragment prepared by the rolling circle amplification method, however, has a specific structure comprising repeating sequences. It is uncertain whether this method can be applied to the circularization of a DNA fragment free from repeating sequences.